CN105934520A - Covalently bound metabolites as biomarkers - Google Patents

Covalently bound metabolites as biomarkers Download PDF

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CN105934520A
CN105934520A CN201580006282.9A CN201580006282A CN105934520A CN 105934520 A CN105934520 A CN 105934520A CN 201580006282 A CN201580006282 A CN 201580006282A CN 105934520 A CN105934520 A CN 105934520A
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disease
macromole
animal
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composition
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CN105934520A8 (en
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韦恩·R·马特森
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Ixcela Inc
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Counterpoint Health Solutions Inc
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Abstract

A method for determining the systems biology state of an animal, which comprises determining levels of small molecules covalently bound to macromolecules (CBSM) in samples from that animal, and using said levels to determine the risk, diagnostic state and progression of disease in that animal. A therapy development method for determining the structure of covalently bound molecules and their precursors and modifying the sources and mechanisms causing such binding to reduce disease risk and progression.

Description

Covalent bond metabolite as biomarker
The present invention relates to for disease and the identification of the biomarker of health status and application.
The present invention is based on following observation: normal processes and lysis all result in the covalency knot of little molecule and macromole Close, and the biological mark guided for disease and therapeutic outcome and treatment that the little molecular composition of these combining forms one class is new Will thing, described mark may utilize series of new techniques and obtains and measure.
In one aspect of the invention, it is provided that a kind of method determining the Animal diseases state such as people, it includes that mensuration is dynamic In thing sample, the micromolecular water with macromole covalent bond (CBSM) is put down, and relatively described level and standard value.
In one embodiment, the little molecule being combined with macromole covalent bond is from enteric microorganism, and it is derived from metabolism The damage of process, Environmental Chemistry or abnormal chemical environment, the mistake between endogenous or inoculating microbe, or one or more above-mentioned sources The interaction of journey.
In another embodiment, described macromole selects free DNA, RNA, protein, carbohydrate compound and sugar In the group of albumen composition.
In yet another embodiment, the choosing of described morbid state free classification of diseases, disease subclassification, progression of disease, disease Risk factor prediction development, treatment specification, the prediction of therapeutic outcome and treatment guide development composition group in.
The method that present invention also offers therapeutic intervention in the disease such as the animal of people, it includes controlling and big point The concentration level of the covalently bound little molecule of son.
In one embodiment, described macromole selects free DNA, RNA, protein, carbohydrate compound and sugar egg In the group of white composition.
In another embodiment, described little molecule is from enteric microorganism, and it is derived from metabolic process, Environmental Chemistry damage Or abnormal chemical environment, the interaction between endogenous or inoculating microbe, or the process in one or more above-mentioned sources.
In one embodiment, described disease is thymopathy, and it selects free depression, schizophrenia and autism Composition group in, degenerative disease, its select free Heng Tingdunshi disease, Alzheimer, Parkinson's disease, mild cognitive impairment, Amyotrophic lateral sclerosis (ALS), uncommon (Freidrich) movement disorder in Delhi, cancer, diabetes and cardiovascular disease composition Group, or selected from metabolism or the birth defect of hereditary.
Present invention also offers a kind of interference method to prevent in the animal such as people with ill danger or to alleviate disease Disease, it covalently bound with macromole little molecule including controlling animal.
In one embodiment, described danger is thymopathy, and it selects free depression, schizophrenia and autism The group of composition, degenerative disease, it selects free Heng Tingdunshi disease, Alzheimer, Parkinson's disease, mild cognitive impairment, flesh Amyotrophic lateral sclerosis disease (ALS), uncommon (Freidrich) movement disorder in Delhi, cancer, diabetes and cardiovascular disease composition Group, or selected from metabolism or the birth defect of hereditary.
The method that present invention also offers the character determining little molecular origin covalently bound with macromole, it includes simulation Biochemical process in such as the animal of people creates and analyzes the synthesis combination of little molecule and macromole.
Present invention also offers the method for modifying gene function in animal, it includes that control is covalently bound with macromole The concentration level of little molecule, is thus increased or decreased the expression of target gene.
In one embodiment, described macromole selects free DNA, RNA, protein, carbohydrate compound and sugar egg In the group of white composition.
In another embodiment, described little molecule is from enteric microorganism, and it is derived from metabolic process, Environmental Chemistry damage Or abnormal chemical environment, the interaction between endogenous or inoculating microbe, or the process in one or more above-mentioned sources.
In yet another embodiment, described gene is relevant to following disease, thymopathy, and it selects free depression, essence God Split disease and autism composition group, degenerative disease, its select free Heng Tingdunshi disease, Alzheimer, Parkinson's disease, Mild cognitive impairment, amyotrophic lateral sclerosis (ALS), uncommon (Freidrich) movement disorder in Delhi, cancer, diabetes and the heart The group of angiopathy composition, or selected from metabolism or the birth defect of hereditary.
Present invention also offers the method finding treatment, comprising:
Identify that a class has the experimenter of equivalent genetic risk factor；
Identify subclass ill and the most ill in this class experimenter；
Identify and DNA, RNA and the difference of the covalently bound molecule of protein, its difference class and subclass to affect system anti- The epigenetic difference that feedback controls；Separate and determine chemistry precursor origin and the structure of covalent bond difference material；And provide or Replace and lose in ill classification or this compound and/or suppression that quantity reduces increase or this of excess in ill classification Plant compound.
In one embodiment, described disease is neurodegenerative diseases, and it selects free Heng Tingdunshi disease, Alzheimer Disease, Parkinson's disease, mild cognitive impairment, amyotrophic lateral sclerosis, Delhi uncommon (Freidrich) movement disorder, cancer, glycosuria The group that sick and cardiovascular disease forms, thymopathy, its free depression of choosing, schizophrenia and the group of autism composition, Or selected from metabolism or the birth defect of hereditary.
Finally the invention provides little molecule covalently bound with macromole, its be used for determining disease risks, diagnostic state, Predictive disease progress and the development for the treatment of.
Further characteristic of the invention and advantage will be by described below and combine accompanying drawing and illustrate, the most identical digital table Show identical part, wherein:
Fig. 1 schematically shows the systems biology feedback net determining the state of organism or individuality, function and risk Network；
Fig. 2 schematically shows preparation flow figure and the mass spectrum thereof of the protein of the little Molecular biomarkers of covalent bond Structural Identification and the position in human plasma；
Fig. 3 A and 3B is collaborative binding curve and LCECA figure (low amplification) of covalent bond curve, wherein Fig. 3 A in blood plasma Show that the collaborative of plasma protein granule extraction from comparison experimenter CC-17 combines little molecule, and Fig. 3 B shows by identical Granule PK digestion after obtain the little molecule of covalent bond；
Fig. 4 A and 4B is the LCECA figure of analysis of control group, it illustrates the PK digestion of blood plasma and for internal surface trimming PK digest blank；
Fig. 5 A-5D shows that comparative control group (CC-17) and HD (CX53) experimenter collaborative combines biomarker LCECA schemes, and described biomarker is by acetonitrile release (left hand view) and the granule of PK digestion ACN precipitation；
Fig. 6 A-6B shows the protein body catapepsis of the plasma A CN precipitation from HD and matched group experimenter LCECA schemes, and it illustrates two kinds of biomarkers of Huntington's disease state feature；
Fig. 7 A-7B is containing KI CAG 140HD mice and the LCECA of the brain tissue extraction component of wild-type mice RNA Figure, it illustrates 8 significant differences in little molecule covalently bound with RNA；
Fig. 8 A-8B is the figure containing the core component from R6/2 mice and the DNA of wild-type mice cerebral tissue, it illustrates 7 differences in covalent bond small-molecule adducts；
Fig. 9 A-C illustrates the enforcement (as a example by indole 3-propanoic acid (I3PA)) of flow chart processes in Fig. 2, prepares covalency In conjunction with the standard substance of little molecule, determine its binding site and the position in LCECA curve thereof；
Fig. 9 A illustrates the preparation of the little molecule of covalent bond of synthesis, and schematically shows by creating and can divide with big Son reaction the method for covalently bound little molecular radical intermediate, create the skill of the standard substance of the little molecule of covalent bond Art；
Fig. 9 B shows the qualification of the fragments of peptides of the covalent bond kynurenine fragment containing oxidation I3PA, and shows Under Fenton reagentia in the synthesis mixing of IPA and angiotensin, the bound site of its initial oxidation product kynurenine (KYA) Point is tyrosine group, and therefore can be detected by LCECA in low-level；And
Fig. 9 C shows and uses synthetically produced covalent bond material to identify covalency knot as standard substance in people experimenter The I3PA closed, and show the comparison of a passage of LCECA curve, the albumen that its display is synthetically produced in HD experimenter The peak height that matter/IPA covalent bond product mates in an array is in matched group, further it is shown that under conditions of biomarker, altogether Valency combines IPA (higher in HD) and is more suitable for making than arbitrary independent compartment significantly with the collaborative ratio combining IPA (lower in HD) For biomarker.
Fig. 1 schematically shows the biochemical interactive network of the individual normal operation of definition.Our disease system Biological concept derives from the hint of this network.Substantially disease is not symptom but regulates and controls unsuccessfully in this network or feedback mistake Lose.Especially for the chronic problem-cardiovascular disease of late onset, neurodegenerative diseases, affective disorder, diabetes, slow Fatigue and other induced disease of immune system, symptom or we are usually said over time, regulate and control unsuccessfully or feed back funeral The disease of the result lost.In Disease epizootic, it is intended under conditions of concentrating on the multiparameter technology finding biomarker Define these networks.
Biomarker, refers to those genes, protein, RNA transcript or the little molecule relevant to disease, and it typically divides Class is: predictive biomarkers, i.e. illustrates the biomarker of disease risks；The biomarker of state, i.e. divides disease Biomarker；The biomarker of progress, i.e. along with the biomarker of progression of disease；And the biological marker of therapeutic outcome Thing, the biomarker i.e. changed along with Results.Defining for these, we increase suggestive therapy's Intervention Strategy now Biomarker.
The research of biomarker is carried out the most at large in concrete " group is learned " compartment (A1-A4 in Fig. 1)- Find gene, gene expression, transcript, protein or collaborative combine little molecule.Seldom carry out phase interaction between " group is learned " compartment Technology and evaluate research and development.Owing to being not wishing to be bound by theory, it is believed that and it is proved these interactions to providing disease The biomarker of disease, therapeutic outcome and treatment development has remarkable effect.The present invention is partially recognized that " group is learned " phase interaction Technology and the data of this interaction are assessed by the disappearance proposing measured.
In biological sample, little Molecular biomarkers and macromole are closely collaborative to be combined.Assess little molecular biosciences mark The technology of will thing (metabolism group) generally uses extraction scheme to remove and to concentrate this collaborative bond material.But, enzyme drives Or the life that normal/abnormal free radical product (such as hydroxyl, epoxide or nitro free radical type or simple similar reaction) drives Thing/biochemical process will cause the macromole covalent bond of these close-connected little molecules and such as protein, DNA or RNA.This Plant the folding/gathering combining the functional and protein that can affect gene expression, enzyme.It is all lysis due to said process With the risk factor of progression of disease, the in principle level of the little molecule of covalent bond and character and the distribution of free little molecule and collaborative In conjunction with little molecular proportion single-gene, transcript and protein or collaborative combine totally preferably reflecting of little molecule and free little molecule Disease or risk factor process.
Referring again to Fig. 1, we have had recognized that this effect, and the method devising assessment feedback link 1,2 and 5, instead Feedback link 1,2 and 5 reflects other feedback procedures 3,4,6 and 7 and enteric microorganism compartment A5.In following non-limiting example Middle by several for description methods for assessment and process.
Embodiment I
Process 1 relate to for blood (blood plasma, leukocyte, platelet, erythrocyte, cell lysis, cracking, whole blood) and His body fluid and covalently bound little Molecular biomarkers of protein of tissue.
Simplest form is, is used for assessing the collaborative use extraction combining little molecule and precipitation blood plasma and its hetero-organization system Standby protein particulate and other macromole (DNA, RNA, carbohydrate compound) are by further chemical digestion or enzymic digestion. Then use metabonomic technology such as Electrochemical Detection liquid chromatograph (LCECA), mass spectrum (MS), the parallel connection of LCEC/LCMS or Tandem compound or nuclear magnetic resonance, NMR (NMR) assess the overview of these preparations.
Shown in the left hand view of this sample preparation methods flow chart in fig. 2.Fig. 3 A-3B shows matched group experimenter's blood In slurry after the acetonitrile extraction component (top) of collaborative binding molecule and the protein body protease K digesting of acetonitrile extraction (bottom) The exemplary embodiments of LCECA collection of illustrative plates.Without in this experiment under derivatising condition use LCECA be only in response to amino acid tyrosine, Tryptophan and methionine or these amino acid whose little dipeptides.In figure, the arrow of bottom is the postdigestive compound of granule, its It is more than aminoacid or little peptide, is also represented by other fragments being combined with the histone amino acid fragment digested.In biological function side Face, these binding compounds will change this critical process in principle, as the performance of enzyme, protein aggregation, cell growth or Cell death.The collection of illustrative plates that a kind of technique and electrochemical array liquid chromatograph (LCECA) subsequently is detected, in accordance with U.S. that applicant is previous State's patent No. 6,194,217 and 6,210,970, it is shown that the response more than 1000 kinds, usually tyrosine, tryptophan or first sulfur ammonia The little dipeptides of acid and little molecule covalently bound with macromole, and respond as above-mentioned amino acid whose adduct.As to according to the facts Executing example, Fig. 4 A-4B shows the human plasma carried out by identical process and the digestion of PK blank.
Fig. 5 A-5D and 6A-6B shows showing of the potential source biomolecule mark of HDvs. matched group in such preparation Example.It should be noted that the collaborative bond material that the comparison experimenter CC17 shown in Fig. 5 A-5B and HD experimenter CX53 extracts LCECA collection of illustrative plates fragment in, there are 3 significant sneak condition biomarkers statistically.But, at Fig. 5 C-5D Illustrate that in the PK digestion pattern of the protein body from these experimenters, biomarker is more notable.Additionally, show at Fig. 5 A-6B In another region of the LCECA collection of illustrative plates gone out, there are two the states lifes illustrating and distinguishing disease and comparison experimenter the most completely Thing mark.
This process can extend to the fractional distillation of protein by size or other modes, is easiest to combine little molecule to determine Specified protein, and more specifically disease or the biomarker of therapeutic outcome are provided or cause the biological marker for the treatment of development Thing.In Fig. 2, the left side of sample preparation flow figure shows this process.
Sample passes through stacked film filter, collected from 1M-300K, 100K-50K-10K molecular weight cut film successively.When When being less than the component of 10K when processing or analyze, directly reflect free metabolite or put down with collaborative binding component in macromole The component of weighing apparatus.The continuous macromolecular components of standard extraction technical finesse precipitated by such as acetonitrile methanol, and divided by collaborative combination The distribution of son analyzes supernatant successively as the function of molecular weight.
Analyzing compared with all collaborative summations combining kind of first group of distributed data, carries potential biomarker Supply more deep understanding.Such as, describe antagonism to press down the relationship part of tryptophan and its primary metabolite kynurenin The reaction of strongly fragrant medicine.But, in the macromolecular components between 300 and 100K, tryptophan is that higher degree is retouched with the relation of kynurenin State；AD blood plasma vs. matched group becomes apparent from the macromolecular components reduced between 100 and 50K of indolepopionic acid, and In 100 to 50K component, the ratio of free material and bond material becomes apparent from.
Obtaining second group of data of macromole precipitation in the case of protein precipitation, protein such as uses trypsin (TP) or protease k (PK) or beta-peptide enzyme or a combination thereof digestion, the component then digested by PK by 10K film or is disappeared by TP The component changed is by 30K film, and direct analysis filtrate.
By introducing boron-doped diamond sensor as last sensor in series, can obtain in electrochemistry array Obtain the analysis of other potential marks, and it be also possible to use LCECA and liquid chromatography-mass spectrography (LCMS) analysis further in parallel, ginseng PCT Application No. PCT/US 13/33918 submitted on March 26th, 2013 according to applicant.Substantially introducing boron doping Buddha's warrior attendant The EC array of stone sensor does not have characteristic indication or not there is in LCEC/LCMS parallel configuration in parallel the extraction mass spectrum of peptide Any response be all little molecule covalently bound with amino acid group.
Tissue DNA, the preparation of RNA
Although the standard that tissue and DNA/RNA can be used to extract prepares scheme, but optimal preparation scheme attempt to protect to The macromole of few chemical damage state.The preparation scheme of tissue includes carrying out macromole dissolving, described process by procedure below Such as ground sample under liquid nitrogen temperature, or make after multigelation in acceptable substrate (such as distilled water or normal saline) With at a high speed " tissue miser " grinder, or circulation high pressure is used again to destroy in suitable substrate.
Embodiment II
The second method of whole blood clinical sample is based on LCECA and the LCMS platform explanation also multiple letter of Quantitative Comparison in parallel Number ability.The crude preparation by using containing DNA is provided by continuous small-bore from the process of blood separation DNA by Series Filtration, its Can with subpackage a kind of component of analytical extraction preparation in order, and directly with HC1 cracking second component so that DNA is broken into alkali Base purine and pyrimido release covalent bond material are as alkali adduct.Then the collection of illustrative plates of two kinds of components is compared to determine that DNA is special Some groups.
For DNA, preliminary study preferably protects the scheme of the histone being combined with DNA.Can be gone by PK digestion selectivity Except histone and as above Digestive system is carried out the little molecule of covalent bond analysis.
Size fractionation scheme can be used all or in isolation to assess RNA component, with assessment and from tRNA, mRNA allochthon Deng the combination of component.Assessment carrys out the macromolecular components of self-organizing, to obtain from the collaborative of above-mentioned metabolism group and covalent bond The distribution of the various protein of compound.Combine metabolism group assessment DNA and RNA component by precipitation/extraction is collaborative, then use P Destroy with AP alkali phosphatase enzyme after 1 endonuclease or P 1 endonuclease or digest with hydrochloric acid or other other weak acid. Under these schemes, the DNA base of such as purification is to the 5' monophosphate (P 1) for digesting for hydrochloric acid or base pair (P 11AP) or base guanine, adenine, cytosine, thymus pyrimidine.As we are reported in article before, look back it Whole collection of illustrative plates of front article be 7 methyl guanines (AnalBiochem.2013May 15；436(2):112-20.doi: 10.1016/j.ab.20I 3.01.035.Epub 2013Feb 12, " A novel method for detecting 7- methyl guanine reveals aberrant methylation levels in Huntington disease", Thomas B,Matson S,Chopra V,Sun L,Sharma S,Hersch S,Rosas HD,Scherzer C, Ferrante R, Matson W), other peaks in response collection of illustrative plates and such as 7 methyl guanines or the alkali of 8 hydroxy guanine The directly modification of base pair is directly related with other molecules, described molecule and base pair or base pair monophosphate or base covalency knot Close.These can be obtained by DNA or RNA course of dissolution, and represents the kind of response sensor inherently, or response is for illustrating not Base pair adduct with chromatographic isolation.
In article before, we there was reported the technology separating RNA and DNA from cerebral tissue, it is assumed that ratio change table Guanine and the targeted approach of 7 methyl guanines in DNA and RNA is developed after showing epigenetic difference.Target analysis is characterized by Obtain guanine and 7 methyl guanines understands signal, which depict in wild type and CAG 140HD mouse model and people is dead Epigenetic change in rear HD and matched group brain.This includes removing and a large amount of operations of LCECA of synergistic combinations.So And, when having we are aware that " chaff interference " as the potential importance of the little molecule of covalent bond, we have reanalysed whole Chromatographic results.
Fig. 7 A-7B and 8A-8B show in KI CAG 140 mice and wild-type mice RNA visibly different 8 kinds its His covalent conjunct agent (methanol component) (Fig. 7 A-7B), and visibly different 11 kinds of other covalency in wild type R6/2 mouse DNA Conjugate (core component) (Fig. 8 A-8B).These other covalent bond adducts use polynary PLS-DA and a test model complete The whole district has divided gene-modified animal and wild type animal.It was additionally observed that the covalent conjunct agent in DNA and RNA has distinguished CAG 140 Late onset HD model and R6/2 initial stage fall ill model.Phenol in this hint HD converts the time of (phenoconversion) Relevant to the individually defined thing that DNA and RNA combines, its in turn suggestive therapy's interference to delay or to prevent to carry the experimenter of HD gene Symptom produce.
Owing to being not wishing to be bound by theory, it is believed that all these materials all may relate to the functional of DNA and RNA, thus Reflect and determine the effect of network in Fig. 1.The function of this network determines the disease outcome of individuality in turn.
Embodiment III
Method for identifying the source of covalent bond material:
The process that many little molecule covalent combine macromole includes by utilizing hydroxyl nitro type free base to carry out attacking wound Build little molecular radical intermediate.We have been prepared for the collaborative position that band is saturated just like the little molecule of kynurenin or indolepopionic acid The various protein of point, RNA and DNA.Using the various variants of Fenton reaction, peroxide/nitrate makes these preparation warps Cross free radical to attack, then carry out as above process.Fig. 4-8 shows the qualification in the source of many responses.We use this side Method identifies the species in PK digestion blood plasma, attacks, as free radical, the compound that indolepopionic acid is formed subsequently with protein bound. The explanation in 9A-C of this process.First, as Fig. 9 A schematically shows, exist in the case of macromole by creating little point The free radical intermediate of son, little molecule combines with macromole (protein or fragments of peptides, DNA, RNA etc.).In this example I Use the reaction of traditional Fenton type to create free radical.In other are applied for, other create the method for free radical intermediate, all As electrochemical oxidation (i.e. for oxyindole) or ultraviolet irradiate (i.e. for the non-electroactive adduct of DNA or RNA) It will be method for optimizing.Second, as shown in Figure 9 B, the material of preparation concentrated and through mass spectrum to determine the ammonia in protein or peptide Basepair binding site in base acid binding site and DNA or RNA.In this example, it is shown that I3PA is combined with tyrosine Product as its reaction intermediate kynurenine.3rd, synthetic standards product are to prepare for the suitable protein of particular studies, Evaluator plasma proteins, human albumin (HSA) in this case.The collaborative bond material of similarly extraction and blood plasma preparation And for differentiating the covalent bond material in human plasma.In this example, lower level association during we identify matched group With combining I3PA and Heng Tingdunshi disease subject, there is the oxidative damage of higher level.
Our analysis shows that the strong of the little Molecular biomarkers of covalent bond (CBSM) identifies number in state and animal It is predicted progress.In the protein of other researchs, the distribution of CBSM makes we conclude that, its measurable depression and spirit point Split the therapeutic outcome of disease, the Phenotype Transition of Heng Tingdunshi disease and mild cognitive impairment be converted into Alzheimer time Between.It is believed that it is suitable for as the biological marker of state, risk, Treatment monitoring and prediction in the range of disease or potential disease Thing.
CBSM can be additionally used in therapeutic intervention and drug development.Reason for this is that, many small molecular phases are to by force altogether Valency combines macromole.Whether heredity or environmental factors induction or the early stage danger shape of h and E factor interaction State, such as the amyotrophic lateral sclerosis in the Gulf War veteran of high incidence or be exposed to insecticide/weeding The Parkinson's disease of the agriculture labourer of agent result in these little molecules and such as DNA or the combination of key protein matter.This combination is anti- Come over to affect the effect (epigenetic) of genome or the functional of enzyme.Such as, the probability of the latter's effect is at dog urinary ammonia Acid approach small molecular is combined with enzyme, affects outbreak or the therapeutic outcome of depression.Owing to being not wishing to be bound by theory, it is believed that this Plant to combine and be probably the reason that in the path relevant from depression, chemical levels is different.Understand which kind of compound is tied with macromole Close and provide design compound or the route of method, with at binding site with without free radical or produce covalently bound chemistry The compound of process replaces.
Understanding alternatively allows the method for designing of compound to change genome with specificity with the covalently bound compound of DNA Function, such as by epigenetic covalent bond close mammary cancer risk gene function.

Claims (13)

1. the method determining Animal diseases state, it includes measuring little molecule covalently bound with macromole in animal sample Level, and relatively described level and standard value.
Method the most according to claim 1, it is characterised in that one or more following characteristics:
A () is wherein said and the covalently bound little molecule of macromole is from enteric microorganism, and it is derived from metabolic process, environmental recovery bonds Learn damage or abnormal chemical environment, the phase interaction between endogenous or inoculating microbe, or the process in one or more above-mentioned sources With；
B () wherein said macromole selects free DNA, RNA, protein, carbohydrate compound and the group of glycoprotein composition In；
C () wherein said morbid state selects the risk factor prediction of free classification of diseases, disease subclassification, progression of disease, disease Development, treatment specification, the prediction of therapeutic outcome and treatment guide development composition group in；And
D () wherein said animal is people.
3. the method being used for the Results of Animal diseases, it includes controlling the dense of little molecule covalently bound with macromole Degree level.
Method the most according to claim 3, it is characterised in that one or more following characteristics:
A () wherein said macromole selects free DNA, RNA, protein, carbohydrate compound and the group of glycoprotein composition In；
B () wherein said little molecule is from enteric microorganism, it is derived from metabolic process, Environmental Chemistry damage or abnormal chemical ring Border, the interaction between endogenous or inoculating microbe, or the process in one or more above-mentioned sources；
C () wherein said disease is thymopathy, it selects free depression, schizophrenia and the group of autism composition, moves back Row disease, it selects free Heng Tingdunshi disease, Alzheimer, Parkinson's disease, mild cognitive impairment, amyotrophic lateral sclerosis Disease, Delhi wish movement disorder, cancer, diabetes and the group of cardiovascular disease composition, or selected from metabolism or the elder generation of hereditary Nature defect；And
D () wherein said animal is people.
5. preventing or improve an interference method for disease in the animal with ill danger, it includes controlling with macromole altogether The little molecule of the animal that valency combines.
Method the most according to claim 5, it is characterised in that one or more following characteristics:
A () wherein said danger is thymopathy, it selects free depression, schizophrenia and the group of autism composition, moves back Row disease, it selects free Heng Tingdunshi disease, Alzheimer, Parkinson's disease, mild cognitive impairment, amyotrophic lateral sclerosis Disease, Delhi wish movement disorder, cancer, diabetes and the group of cardiovascular disease composition, or selected from metabolism or the elder generation of hereditary Nature defect；And
B () wherein said animal is people.
7. the method determining the character of little molecular origin covalently bound with macromole, it is included in animal with simulation raw Change process creates and analyzes the synthesis combination of little molecule and macromole.
Method the most according to claim 7, wherein said animal comprises people.
9. a method for modifying gene function in animal, it includes controlling the concentration with the covalently bound little molecule of macromole Level, is thus increased or decreased the expression of target gene.
Method the most according to claim 9, it is characterised in that one or more following characteristics:
A () wherein said macromole selects free DNA, RNA, protein, carbohydrate compound and the group of glycoprotein composition In；
B () wherein said little molecule is from enteric microorganism, it is derived from metabolic process, Environmental Chemistry damage or abnormal chemical ring Border, the interaction of the process between endogenous or inoculating microbe, or one or more above-mentioned sources；
C () wherein said gene is relevant to following disease, thymopathy, and it selects free depression, schizophrenia and solitarily Disease composition group, degenerative disease, its select free Heng Tingdunshi disease, Alzheimer, Parkinson's disease, mild cognitive impairment, Amyotrophic lateral sclerosis, Delhi wish movement disorder, cancer, diabetes and the group of cardiovascular disease composition, or selected from metabolism or The birth defect of hereditary；And
D () wherein said animal is people.
11. 1 kinds of methods treating discovery, comprising:
Identify that a class has the experimenter of equivalent genetic risk factor；
Identify the subclass that this apoplexy due to endogenous wind is ill and the most ill；
Identifying and the difference in DNA, RNA and protein covalent bond molecule, its difference classification and subclass also affect system feedback Epigenetic difference in control；
Separate and determine that chemistry precursor origin and covalent bond differentiate the structure of material；And
Thering is provided or replacing loss or quantity reduce in ill classification this compound and/or suppression increases in ill classification Or this compound of excess.
The method of 12. discoveries according to claim 11, wherein said disease is:
Neurodegenerative diseases, it selects free Heng Tingdunshi disease, Alzheimer, Parkinson's disease, mild cognitive impairment, flesh to wither Contracting lateral schlerosis, Delhi wish movement disorder, cancer, diabetes and the group of cardiovascular disease composition, and thymopathy, it is selected from The group being made up of depression, schizophrenia and autism, or selected from metabolism or the birth defect of hereditary.
13. with the covalently bound little molecule of macromole, it is used for determining disease risks, diagnostic state, predictive disease progress and controls The development treated.
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